Codeine, morphine, and heroin are the three most commonly abused drugs among opiates. Codeine and morphine are natural materials derived from opium while heroin is a synthetic chemical made from acetylation of morphine. In human the main metabolic pathway of these opiates is glucuronidation which occurs in liver microsomes. The major metabolite of codeine is codeine-6.beta.-glucuronide while the major metabolite of morphine and heroin is morphine-3.beta.-glucuronide. Unmetabolized codeine, morphine, and heroin constitute only a very small amount in biological samples. Since morphine-3.beta.-glucuronide is also the major metabolite of heroin, it is apparent that deacetylation of heroin occurs before glucuronidation. In fact the identification of a minor but very characteristic metabolite called 6-monoacetylmorphine has been established as evidence of heroin use. This 6-monoacetylmorphine is the result of the deacetylation of heroin. Another important aspect of morphine and heroin metabolism is the formation of a second glucuronide metabolite, morphine-6.beta.-glucuronide, which, even though usually found in smaller amount compared to morphine-3.beta.-glucuronide, has been shown to be as potent as morphine itself.
Deuterated codeines such as codeine-d3 and codeine-d6 are commercially available and have been used as internal standards for Gas chromatography-Mass spectrometry (GC-MS) analysis of codeine in biological samples. In the process of analysis, deuterated codeine is added in a known amount to samples which are then undergo sample preparation processes before analysis. The use of deuterated codeine as internal standard in GC-MS analysis takes advantage of the fact that any losses of codeine in extraction, derivatization, and transfering processes will also result in similar losses of deuterated codeine. The analysis of codeine using deuterated codeine as internal standard is, therefore, more reliable than the same analysis using codeine analogs as internal standards. Unmetabolized codeine in biological samples are usually in small amount compared to the major metabolite codeine-6.beta.-glucuronide. To increase the sensitivity of GC-MS analysis of codeine, samples usually undergo a hydrolysis step to convert codeine-6.beta.-glucuronide to codeine. This hydrolysis step is very important in GC-MS analysis of opiates and is usually carried out by either acid or base or enzyme. Cares have to be taken in hydrolysis because if the enzyme is not good or if the acid or base is not strong enough, hydrolysis will be incomplete. If the conditions are too harsh, opiates will decompose. Thus if codeine-6.beta.-glucuronide is not completely hydrolysed to codeine, the total codeine concentration in samples will be low and the interpretation of the result can be false negative. It would be desirable to have available deuterated codeine-6.beta.-glucuronides to be used as internal standards for GC-MS analysis of codeine, then the incomplete hydrolysis of codeine-6.beta.-glucuronide will likely result in the same incomplete hydrolysis of deuterated codeine-6.beta.-glucuronide. The GC-MS analysis of codeine using deuterated codeine-6.beta.-glucuronides as internal standards, therefore, would be more reliable than the same analysis using deuterated codeines as internal standards. The availability of deuterated codeine-6.beta.-glucuronides also allows direct analysis of codeine-6.beta.-glucuronide in such techniques that do not require sample hydrolysis as Liquid chromatography-Mass spectrometry (LC-MS). So far the analysis of codeine-6.beta.-glucuronide by LCMS using deuterated codeine-6.beta.-glucuronides has not been reported.
Deuterated morphine such as morphine-d3 is available and is currently used as internal standard for GC-MS analysis of morphine in biological samples of morphine and heroin users. Samples are usually spiked with a known amount of morphine-d3 before subjected to a hydrolysis step to convert all morphine glucuronides (morphine-3.beta.-glucuronide constitutes the highest percentage) and acetylated morphines (very small percentage) to morphine. Again, an incomplete hydrolysis will likely result in lower value for total morphine. Thus it is also desirable to have available deuterated morphine-3.beta.-glucuronides to be used as internal standards for GC-MS analysis of morphine or heroin uses.
Current advances of mass spectrometry already allow analysis of glucuronide conjugates directly. The availability of deuterated codeine-6.beta.-glucuronides and deuterated morphine-3.beta.-glucuronides will allow the direct MS analysis of codeine, morphine, and heroin major metabolites without hydrolysis. The analysis will be simple and the results are more accurate. The analysis can also allow simultaneous determination of unmetabolized codeine, morphine, heroin, and even the characteristic heroin metabolite 6-monoacetylmorphine using the available respective deuterated internal standards.
It has been shown that morphine-6.beta.-glucuronide, the minor metabolite of morphine and heroin in human, is as potent as the parent compound itself The concentration of this metabolite is carefully monitored in subjects given doses of morphine or heroin. Its concentration has been monitored independent of morphine-3.beta.-glucuronide, the inactive metabolite, using High pressure liquid chromatography (HPLC) with either ultraviolet detector or electrochemical detector or fluorometric detector. The current GC-MS analysis method for morphine cannot distinguish between morphine-6.beta.-glucuronide and morphine-3.beta.-glucuronide. The availability of deuterated morphine-6.beta.-glucuronides and deuterated morphine-3.beta.-glucuronides will allow a more sensitive monitoring of both morphine-6.beta.-glucuronide and morphine-3.beta.-glucuronide by methods that do not require sample hydrolysis such as HPLC using MS as detector (usually called LC-MS).
The potency of morphine-6.beta.-glucuronide has led to the manufacture and use of this material as therapeutic drug. The availability of deuterated morphine-6.beta.-glucuronides also allows metabolism studies of this drug even in subjects previously taken medications containing codeine, morphine, or heroin.
The syntheses of codeine-6.beta.-glucuronide, morphine-3.beta.-glucuronide and morphine-6.beta.-glucuronide are documented but there are no known synthesis of deuterated codeine-6.beta.-glucuronide, morphine-3.beta.-glucuronides and morphine-6.beta.-glucuronides. To be useful deuterated opiate glucuronides have to be able to transform to deuterated opiates upon hydrolysis. The generated deuterated opiates have to be stable to all sample preparation processes (hydrolysis, extraction, concentration, derivatization). That also means that none of deuterated opiate glucuronides is converted to undeuterated opiates which will apparently increase the concentration of opiates in the analysis.